Time-Restricted Salutary Effects of Blood Flow Restoration on Venous Thrombosis and Vein Wall Injury in Mouse and Human Subjects

Abstract

Background: Up to 50% of patients with proximal deep vein thrombosis (DVT) will develop the postthrombotic syndrome characterized by limb swelling and discomfort, hyperpigmentation, skin ulcers, and impaired quality of life. Although catheter-based interventions enabling the restoration of blood flow (RBF) have demonstrated little benefit on postthrombotic syndrome, the impact on the acuity of the thrombus and mechanisms underlying this finding remain obscure. In experimental and clinical studies, we examined whether RBF has a restricted time window for improving DVT resolution.

Methods: First, experimental stasis DVT was generated in C57/BL6 mice (n=291) by inferior vena cava ligation. To promote RBF, mice underwent mechanical deligation with or without intravenous recombinant tissue plasminogen activator administered 2 days after deligation. RBF was assessed over time by ultrasonography and intravital microscopy. Resected thrombosed inferior vena cava specimens underwent thrombus and vein wall histological and gene expression assays. Next, in a clinical study, we conducted a post hoc analysis of the ATTRACT (Acute Venous Thrombosis: Thrombus Removal with Adjunctive Catheter-Directed Thrombolysis) pharmacomechanical catheter-directed thrombolysis (PCDT) trial (NCT00790335) to assess the effects of PCDT on Venous Insufficiency Epidemiological and Economic Study quality-of-life and Villalta scores for specific symptom-onset-to-randomization timeframes.

Results: Mice that developed RBF by day 4, but not later, exhibited reduced day 8 thrombus burden parameters and reduced day 8 vein wall fibrosis and inflammation, compared with controls. In mice without RBF, recombinant tissue plasminogen activator administered at day 4, but not later, reduced day 8 thrombus burden and vein wall fibrosis. It is notable that, in mice already exhibiting RBF by day 4, recombinant tissue plasminogen activator administration did not further reduce thrombus burden or vein wall fibrosis. In the ATTRACT trial, patients receiving PCDT in an intermediate symptom-onset-to-randomization timeframe of 4 to 8 days demonstrated maximal benefits in Venous Insufficiency Epidemiological and Economic Study quality-of-life and Villalta scores (between-group difference=8.41 and 1.68, respectively, P<0.001 versus patients not receiving PCDT). PCDT did not improve postthrombotic syndrome scores for patients having a symptom-onset-to-randomization time of <4 days or >8 days.

Conclusions: Taken together, these data illustrate that, within a restricted therapeutic window, RBF improves DVT resolution, and PCDT may improve clinical outcomes. Further studies are warranted to examine the value of time-restricted RBF strategies to reduce postthrombotic syndrome in patients with DVT.

Keywords: fibrinolysis; inflammation; postthrombotic syndrome; thrombectomy; venous thrombosis.

Figures

Figure 1.. A murine IVC DVT ligation/de-ligation model that enables time-dependent restoration of blood flow (RBF) of occlusive DVT.

(A) Experimental flowchart demonstrating surgical IVC knot de-ligation performed at day 2 or day 4, and assessment of RBF using ultrasound (US) or intravital microscopy (IVM). (B) Schematic illustration of initial complete ligation of the IVC on day 0 using a spacer. At day 2 or day 4, IVC knot de-ligation and spacer removal was performed to spur RBF. (C) In vivo assessment of IVC DVT anatomy and blood flow at day 8 in: control mice without DVT; mice with occlusive DVT; and mice with DVT and RBF. Representative IVC images in each group as shown by IVM venography and US. In the RBF group (right images), FITC-dextran signal is seen in between the thrombus and vein wall on IVM (yellow arrows) and on US (yellow arrows). Doppler and pulse wave US reveals opposing blood flow direction in the abdominal aorta (Ao) and IVC, as expected. Absent IVC blood flow on US indicated occlusive DVT, without RBF. Flanking IVC “bridging” collaterals (Co) were also noted in some mice with occlusive DVT. The red solid line demarcates the thrombus. The yellow, red and blue dotted lines/circles indicate the IVC, aorta and collateral vein, respectively. The yellow arrows indicate IVC blood flow. (D) Contingency table comparing ultrasound and venography (reference standard) based on the IVC RBF status. (E) Diagnostic performance of ultrasound compared to venography. Ultrasound showed high sensitivity (86.0%), specificity (96.2%), and positive predictive value (99.0%) for assessing RBF in IVC DVT, as compared to IVM venography. Ultrasound also showed good agreement with venography (κ=0.72). Abbreviations: IVC, inferior vena cava; DVT, deep vein thrombosis; US, ultrasound; IVM, intravital microscopy; H, head; T, tail; Co, collateral vein; R, right; L, left; Ao, abdominal aorta; PV: predictive value.

Figure 2.. Temporal assessment of mechanical de-ligation, RBF, and venous thrombus burden at day 8.

(A) Experimental flowchart for mice undergoing day 2 IVC DVT de-ligation, followed by RBF assessment using serial US and then venography prior to sacrifice at day 8. (B) The percentage of mice establishing RBF (%) increased steadily after day 2 de-ligation. RBF that developed by day 4 was defined as early RBF, and RBF that developed after day 4 was defined as late RBF. The day 2 de-ligation group demonstrated higher RBF rates starting at 2 days post de-ligation, compared to sham de-ligation; N.S., p>0.05; ***p<0.001; Fisher’s exact test for the comparison of RBF at each timepoint. (C-F) Thrombus burden measures in mice undergoing day 2 de-ligation, as a function of RBF status confirmed by US. N.S., p>0.05; *p<0.05; **p<0.01; one-way ANOVA and Tukey’s test for normally distributed data; Kruskal-Wallis and Dunn’s test for not normally distributed data; n=3-11 animals per group. (G) Experimental flowchart for mice undergoing day 4 IVC DVT de-ligation. (H) The RBF% through DVT increased gradually over time after day 4 de-ligation. The day 4 de-ligation group demonstrated higher RBF rates starting at 4 days post de-ligation, compared to sham de-ligation; N.S., p>0.05; *p=0.052; **p<0.01; Fisher’s exact test. (I-L) Thrombus burden measures in mice undergoing day 4 de-ligation, as a function of RBF status confirmed by US. N.S., p>0.05; one-way ANOVA and Tukey’s for normally distributed data; Kruskal-Wallis and Dunn’s test for not normally distributed data; n=7-10 animals per group. Abbreviations: IVC, inferior vena cava; DVT, deep venous thrombus; h, hour; US, ultrasound; IVM, intravital microscopy; de-li: de-ligation; D, day; RBF, restored blood flow; No., number.

Figure 3.. Temporal and dose effects of exogenous rtPA therapy on venous thrombus burden at day 8.

(A) Experimental flowchart for mice undergoing day 2 IVC DVT de-ligation and then day 4 rtPA reperfusion therapy. RBF assessment by US was performed at day 4 prior to rtPA administration, and then at day 6 and day 8, followed by sacrifice at day 8 for thrombus burden. (B) High-dose rtPA non-significantly increased the RBF% from 55.6% to 76.9% at day 6; minimal change in the RBF% was evident in the low-dose rtPA group or control group. N.S., p>0.05; Fisher’s exact test for the comparison of RBF at each timepoint. (C-F) Effects of day 4 rtPA infusion on day 8 thrombus burden, as a function of rtPA dose and RBF status. N.S., p>0.05; *p<0.05; **p<0.01; ***p<0.001; two-way ANOVA and Tukey’s test; n=4–23 animals per group. (G) Experimental flowchart for mice undergoing day 4 IVC DVT de-ligation and then day 6 rtPA therapy. (H) RBF% through DVT after day 6 rtPA. High-dose rtPA did not increase the rate of mice achieving RBF. N.S., p>0.05; Fisher’s exact test. (I-L) Effects of day 6 rtPA infusion on day 8 thrombus burden, as a function of rtPA dose and RBF status. N.S, p>0.05; two-way ANOVA and Tukey’s test; n=5-14 animals per group. Abbreviations: D, day; de-li, de-ligation; sac, sacrifice; RBF, restoration of blood flow; IVC, inferior vena cava; DVT, deep venous thrombosis; US, ultrasound; h, hour; rtPA, recombinant tissue plasminogen activator.

Figure 4.. Temporal assessment of mechanical de-ligation +/- pharmacological rtPA-induced RBF on vein wall fibrosis at day 8.

(A) Schematic illustration of histological section assessment from the infrarenal IVC to iliac bifurcation every 1.2 mm. (B) Representative images of IVC VWCT (vein wall fibrosis) using Carstairs’ and picrosirius red (PSR) stains. Scale bar, 30 μm. (C) Assessment of IVC VWCT at day 8 as a function of RBF status at day 4. The black dotted line shows the mean VWCT (9.4 mm) of the normal IVC. **p<0.01; ***p<0.001; one-way ANOVA and Tukey’s; n=7–10 animals per group. (D) Differences in IVC VWCT at day 8 as function of IVC location and RBF status were further analyzed by two-way ANOVA, followed by Tukey’s. N.S, p>0.05; ***p<0.001, n=7-10 animals per group. (E) Assessment of IVC VWCT at day 8 as a function of rtPA therapy and RBF status. In mice that were already RBF+ at day 4, rtPA did not further reduce the mean VWCT at day 8. N.S, p>0.05; two-way ANOVA and Tukey’s test; n=4-15 animals per group. (F) Day 8 VWCT as function of IVC location and rtPA dose at day 4, analyzed by two-way ANOVA and Tukey’s test. No rtPA groups re-displayed from Figure 4D. N.S, p>0.05; **p<0.01; ***p<0.001; n=4-15 animals per group. (G-J) Correlative trends emerged between the day 8 group average of VWCT and thrombus burden parameters across the 7 groups, with borderline significant associations with thrombus mass, weight and width (n=4-15 for VWCT, and n=4-23 for thrombus burden; r=0.75-0.77, p=0.051-0.066), but not for thrombus length (n=4-23, r=0.23, p=0.61), as analyzed by Spearman’s rank correlation. Scar bar indicates standard error. (K) Vein wall fibrosis measurements in a subgroup of mice sacrificed at day 4 or day 8, as a function of RBF status. Day 8 data re-displayed from Figure 4E. N.S., p>0.05; ***p<0.001; two-way ANOVA with Tukey’s test; n=4–15 animals per group. Abbreviations: VWCT, vein wall collagen thickness; IVC, inferior vena cava; PSR, picrosirius red staining; de-li, de-ligation; d, day; RBF, restored blood flow; sac, sacrifice; rtPA, recombinant tissue plasminogen activator; h, high; avg., average.

Figure 5.. Effects of RBF on inflammatory and collagen synthetic mediators in the vein wall following venous thrombosis.

(A) mRNA transcript levels of genes of interest in the IVC wall detected by qRT-PCR at day 8, as a function of RBF status at day 4. Relative fold change compared to the d4 RBF+ group, set at 1.0 (dotted line). Data are presented as mean ± SEM. *p<0.05; **p<0.01; unpaired Students’ t-test for normally distributed data; Mann-Whitney test for not normally distributed data; n=5–9 animals per group. (B) Representative images of IVC wall F4/80+ macrophages and FSP1+ fibroblasts at day 8. Scale bars, 50 μm. (C-D) Day 8 vein wall F4/80+ macrophage and FSP1+ fibroblast cell numbers per 5 HPFs, stratified by RBF status at day 4. ***p<0.001; **p<0.01; unpaired Students’ t-test for normally distributed data; Mann-Whitney test for not normally distributed data; n=7–8 mice per group. (E) The relationship between the day 8 VWCT and vein wall F4/80+ macrophages was not significant (p=0.24). (F) A significant relationship was present between the day 8 VWCT and vein wall FSP1+ fibroblasts (p=0.022). Relationships analyzed by multiple linear regression with adjustment for groups. Abbreviations: D, day; IVC, inferior vena cava; RBF, restoration of blood flow; FSP1, fibroblast-specific protein 1; TNF, tumor necrosis factor; IL, interleukin; MMP, matrix metalloproteinase; Cat B, cathepsin B; TGF, transforming growth factor; uPA, urokinase-type plasminogen activator; PAI-1, plasminogen activator inhibitor type 1; PSR, Picrosirius red; Cars, Carstairs’ IHC, immunohistochemistry; T, thrombus; W, vein wall; L, lumen; VWCT, vein wall collagen thickness; HPF, high-power field.

Figure 6.. Analysis of different timepoints and their effects of PCDT efficacy for the VEINES-QoL score in the ATTRACT trial.

ATTRACT patients underwent a non-prespecified subgroup analysis categorized as three subgroups based on their symptom-onset-to-randomization timepoint. The mean VEINES-QoL score was estimated by piecewise linear-regression growth-curve models using all available visit assessments from baseline to 24 months, with adjustment for the location of DVT (iliofemoral vs. femoral-popliteal DVT), clinical center and baseline covariates (age, sex, BMI), using an intention-to-treat analysis. To account for multiple testing, a Bonferroni adjusted p-value ≤0.0056 was considered as statistically significant. The benefit of PCDT therapy on VEINES-QoL score was significantly higher in the intermediate SOR timeframe compared to control group. The maximum benefit was evident in the SOR 4–8 day timeframe (p=0.00038). The interaction between treatments × SOR times (G1 vs. G2 vs. G3) was also significant for intermediate SOR 4–8 days (p=0.020). The interaction between treatments × SOR times from G2 vs. the combined G1+G3 group was even more significant for the intermediate SOR 4–8 days (p=0.0089). Abbreviations: SOR, symptom-onset-to-randomization (in days); No, number; PCDT, pharmacomechanical catheter-directed thrombolysis; VEINES-QoL, Venous Insufficiency Epidemiological and Economic Study-Quality of Life; CI, confidence interval; Tmts, treatments; G, group.